The Bordetella pertussis gene encoding superoxide dismutase (SOD), termed sodB, was cloned. The nucleotide sequence of sodB predicted a 21-kDa protein with homology to iron-containing SODs. Examination of SOD activity gels suggests that B. pertussis extracts contain a single SOD that utilizes Fe³⁺ as its prosthetic group. A SOD-deficient mutant, DD25, was obtained by insertional inactivation of sodB in B. pertussis. DD25 was more sensitive to killing by the superoxide-generating compound paraquat than the parental strain BP339. Both strains survived equally in human polymorphonuclear leukocytes (PMN). Crude extracts of B. pertussis contain a single catalase. B. pertussis katA was cloned and the nucleotide sequence predicted a 55-kDa protein that shared homology with heme-containing catalases. Analysis of the nucleotide sequence upstream of katA revealed the presence of a copy of IS481, a B. pertussis-specific insertion sequence. The start site of transcription of katA was mapped to a T residue in IS481 by primer extension analysis. A catalase-deficient strain of B. pertussis, DD900, was constructed and was more sensitive to H₂O₂ killing than the parental strain BP339. However, there was no difference in the ability of either strain to survive in PMN. The genes encoding pertussis toxin (ptx) and adenylate cyclase toxin (cya) are positively regulated in B. pertussis by the bvgAS locus. However, ptx::lacZ and cya::lacZ transcriptional fusions in E. coli cannot be activated by bvgAS in trans. This suggests that an additional factor(s) is required for transcription of these genes. A gene encoding a Bvg accessory factor (BAF) was identified by it's ability to activate ptx::lacZ in the presence of bvgAS. The expression of ptx::lacZ was decreased by the addition of 40 mM MgSO₄, a compound that also modulates ptx expression in B. pertussis. An additional copy of baf was integrated into the chromosome of BC75, a B. pertussis mutant that expresses low levels of ptx and cya. The defect in BC75 was partially complemented in the cointegrate strain. The gene encoding BAF was localized, sequenced, and found to produce a 28-kDa protein. BAF appears to be a new kind (class) of transcriptional regulator.

The Bordetella pertussis gene encoding superoxide dismutase (SOD), termed sodB, was cloned. The nucleotide sequence of sodB predicted a 21-kDa protein with homology to iron-containing SODs. Examination of SOD activity gels suggests that B. pertussis extracts contain a single SOD that utilizes Fe³⁺ as its prosthetic group. A SOD-deficient mutant, DD25, was obtained by insertional inactivation of sodB in B. pertussis. DD25 was more sensitive to killing by the superoxide-generating compound paraquat than the parental strain BP339. Both strains survived equally in human polymorphonuclear leukocytes (PMN). Crude extracts of B. pertussis contain a single catalase. B. pertussis katA was cloned and the nucleotide sequence predicted a 55-kDa protein that shared homology with heme-containing catalases. Analysis of the nucleotide sequence upstream of katA revealed the presence of a copy of IS481, a B. pertussis-specific insertion sequence. The start site of transcription of katA was mapped to a T residue in IS481 by primer extension analysis. A catalase-deficient strain of B. pertussis, DD900, was constructed and was more sensitive to H₂O₂ killing than the parental strain BP339. However, there was no difference in the ability of either strain to survive in PMN. The genes encoding pertussis toxin (ptx) and adenylate cyclase toxin (cya) are positively regulated in B. pertussis by the bvgAS locus. However, ptx::lacZ and cya::lacZ transcriptional fusions in E. coli cannot be activated by bvgAS in trans. This suggests that an additional factor(s) is required for transcription of these genes. A gene encoding a Bvg accessory factor (BAF) was identified by it's ability to activate ptx::lacZ in the presence of bvgAS. The expression of ptx::lacZ was decreased by the addition of 40 mM MgSO₄, a compound that also modulates ptx expression in B. pertussis. An additional copy of baf was integrated into the chromosome of BC75, a B. pertussis mutant that expresses low levels of ptx and cya. The defect in BC75 was partially complemented in the cointegrate strain. The gene encoding BAF was localized, sequenced, and found to produce a 28-kDa protein. BAF appears to be a new kind (class) of transcriptional regulator.

en_US

dc.type

text

en_US

dc.type

Dissertation-Reproduction (electronic)

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thesis.degree.name

Ph.D.

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thesis.degree.level

doctoral

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thesis.degree.discipline

Microbiology and Immunology

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thesis.degree.discipline

Graduate College

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thesis.degree.grantor

University of Arizona

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dc.contributor.chair

Friedman, Richard L.

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dc.contributor.committeemember

Ito, Junetsu

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dc.contributor.committeemember

Bernstein, Harris

en_US

dc.contributor.committeemember

Joens, Lynn

en_US

dc.contributor.committeemember

Komm, Barry

en_US

dc.identifier.proquest

9502605

en_US

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